With the explosion of techniques for the synthesis, analysis and manipulation of nucleic acids, numerous new opportunities have arisen in diagnostics and therapeutics. In research there is substantial interest in being able to identify DNA sequences, which may be associated with specific organisms, alleles, mutations, and the like, to understand particular genetic processes, to identify diseases, for forensic medicine, etc. Also, for many purposes, one may wish to modulate the activity of a particular gene, so as to identify the function of a particular gene, the effect of changes in its cellular concentration on the function of the cell, or other cellular characteristic. In therapeutics, one may wish to inhibit the proliferation of cells, such as bacterial, fungal and chlamydia cells, which may act as pathogens, of viruses, of mammalian cells, where proliferation results in adverse effects on the host, or other situation. In vivo, one may provide for reversible or irreversible knock out, so that information can be developed on the development of a fetus, or the effect on the organism of reduced levels of one or more genetic products.
In a number of seminal papers, Peter Dervan's group has shown that oligomers of nitrogen heterocycles can be used to bind to dsDNA. It has been shown that there is specificity in that G/C is complemented by N-methyl imidazole (Im)/ N-methyl pyrrole (Py), C/G is complemented by Py/Im, A/T and T/A are redundantly complemented by Py/Py. In effect, N-methyl imidazole tends to be associated with guanosine, while N-methyl pyrrole is associated with cytosine, adenine, and thymidine. By providing for two chains of the heterocycles, as 1 or 2 molecules, a 2:1 complex with dsDNA is formed, with the two chains of the oligomer antiparallel, where G/C pairs have Im/Py in juxtaposition, C/G pairs have Py/Im, and T/A pairs have Py/Py in juxtaposition. The heterocycle oligomers are joined by amide carbamyl) groups, where the NH may participate in hydrogen bonding with nitrogen unpaired electrons, particularly of adenine. While the complexes were of substantial interest, the binding affinities for the most part were less than about 10.sup.6 M.sup.-1. Furthermore, the discrimination between a target DNA sequence, and one involving a mismatch was frequently not better than about two-fold. Therefore, for many purposes, the complexes had limited utility.
Improvements in affinity were shown for a cyclic dimer, where the two oligomers were joined at their ends by .gamma.-aminobutyric acid, where the affinity was shown to be enhanced to about 10.sup.9 M.sup.-1. However, the difference in affinity between the target sequence and single-base mismatch sequences were less than three-fold difference for three different single-base mismatch sequences. This would severely limit the applications for the compound in the presence of a large amount of naturally occurring dsDNA.
Also, for many applications, one wishes to be able to use the sequences with viable cells. There was no showing that these oligomers would be capable of being transported across a cellular membrane to the nucleus and, upon successful transport to the nucleus, they could bind to the chromosomal DNA, where the chromosomal DNA is present as nucleosomes.
Relevant Literature
Wade et al., J.AM.CHEM.SOC., 1992, 114, 8783-8794; Mrkish et al., PROC.NATL.ACAD.SCI. USA, 1992, 89, 7856-7590; Mrkish and Dervan, J.AM.CHEM.SOC., 1993, 115, 2572-2576; Wade et al., Biochemistry, 1993, 32, 11385-11389; Mrkish and Dervan, J.AM.CHEM.SOC., 1993, 115, 9892-9899; Dwyer et al., J.AM.CHEM.SOC., 1993, 115, 9900-9906; Mrkish and Dervan, J.AM.CHEM.SOC., 1994, 116, 3663-3664; Mrkish et al, J.AM.CHEM.SOC., 1994, 116, 7983-7988; Mrkish and Dervan J.AM.CHEM.SOC., 1995, 117, 3325-3332; Cho et al., PROC.NATL.ACAD.SCI. USA, 1995, 92, 10389-10392; Geierstanger, Nature Structural Biology, 1996, 3, 321-324; Parks et al., J.AM.CHEM.SOC., 1996, 118, 6147-6152; Parks et al., J.AM.CHEM.SOC., 1996, 118, 6153-6159; Baird and Dervan, J.AM.CHEM.SOC., 1996, 118, 6141-6146; Swalley et al., J.AM.CHEM.SOC., 1996, 118, 8198-8206; Trauger et al., J.AM.CHEM.SOC., 1996, 118, 6160-6166; Szewczyk et al., J.AM.CHEM.SOC., 1996, 118, 6778-6779; Trauger et al., Chemistry & Biology, 1996, 3, 369-377; Trauger et al., Nature, 1996, 382, 559-561; Kelly et al., PROC.NATL.ACAD.SCI. USA, 1996, 93, 6981-6985; Szewczyk et al., ANGEW.CHEM.INT.ED.ENGL., 1996, 35, 1487-1489; Pilch et al., PROC.NATL.ACAD.SCI. USA, 1996, 93, 8306-8311; White et al., Biochemistry, 1996, 35, 12532-12537.